Phosphorylated Akt (Ser 473) was obtained from Cell Signaling Technology (Danvers, INCB28060 mouse MA). Vimentin was obtained
from BD Biosciences (Franklin Lakes, NJ). α-Tubulin and phalloidin-TRITC were purchased from Sigma (St. Louis, MO). Pharmacological Treatments OSCC cells were plated at 2–2.5 × 105 cells/well in 6- or 12-well plates in DMEM containing 10% FBS and incubated for 24 h. The medium was then changed to DMEM with 0.1% FBS, and the cells were incubated overnight. After overnight incubation, cells were treated with PIA dissolved in DMSO (5 μM) for 12 h (in vitro migration assay) or 24 h (other experiments). In all experiments, DMSO added to control samples had no effect on Akt activity. RT-PCR mRNA was purified from the cells using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommended protocol. Two μg RNA was added to RT-PCR reactions containing primers at a concentration of 0.5 μM. After a 42°C/60-min reverse transcription step, 30 cycles of PCR amplification were performed at 94°C for 30 sec, 58°C for 50 sec, and 72°C for 50 sec. PCR products were run on 1.5% agarose gels for identification. Primers used were 5′-TCC CAT CAG CTG CCCAGA AA-3′ and 5′-TGA CTC CTG TGT TCC TGT TA-3′ for E-cadherin, 5′-AAG CAG GAG TCC ACT GAG
TA-3′ and 5′-GTA TCA ACC AGA GGG AGT GA-3′ for Vimentin, 5′-GGG CAG GTA TGG AGA
GGA AGA-3′ and 5′-TTC TTC TGC GCT ACT selleck chemicals llc GCT GCG-3′ for Snail, 5′-TTC CTG GGC TAC GAC CAT AC-3′ and 5′-GCC TTG AGT GCT CGA TAA-3′ for Sip1, 5′-GGA GTC CGC AGT CTT ACG AG-3′ and 5′-TCT GGA GGA CCT GGT AGA GG-3′ for Twist, 5′-GCT GAT TTG ATG GAG TTG GA-3′ and 5′-GCT ACT TGT TCT TGA GTG AA-3′ for β-catenin, and 5′-GAA GGT GAA GGT CGG AGT C-3′ and 5′-CAA AGT TGT CAT GGA TGA CC-3′ for GAPDH. Analysis of the E-cadherin promoter by CB-839 concentration Methylation specific-PCR (MS-PCR) Methylation status of the CpG sites in the E-cadherin promoter region was analyzed based on the principle that bisulfite modification of the genomic DNA would convert unmethylated cytosine residues to uracil, whereas methylated cytosine is resistant to HSP90 the treatment. Bisulfite modification and MS-PCR were carried out as described [17, 18]. Modified DNA was amplified using primers specific for the methylated sequence (5′-TTA GGT TAG AGG GTT ATC GCG T-3′ and 5′-TAA CTA AAA ATT CAC CTA CCG AC-3′ and for the unmethylated sequence (5′-TAA TTT TAG GTT AGA GGG TTA TTG T-3′ and 5′-CAC AAC CAA TCA ACA ACA CA-3′). 35 cycles of PCR amplification were performed at 94°C for 30 sec, 56°C for 30 sec, and 72°C for 30 sec. PCR products were run on 2% agarose gels for identification.